Elecrophoresis involves the separation of charged macromolecular species in a carrier medium in an electric field. Now electrophoresis involves migration of those charged molecular species through a porous gel under an applied electric field. Most widely used gel is polyacrylamide, typically cross linked with a small amount of bis-acrylamide; however, other gels may also be used.
Gel electrophoresis separations may be on the basis of molecular weight, pH or a combination of the two. Suitable gels and techniques for each type of separation are known. Electrophoresis separation is more widely used for separation of mixture of macromolecules according to molecular weight.
Electrophoresis is a particularly desirable tool for separation or fractionation of macromolecular mixtures in which the molecular weights of the species present are from about 10,000 to about 1 million. Gel electrophoresis is a particularly suitable technique for fractionating protein mixtures, including gene mixtures used in genetic studies.
While electrophoresis has some limited use as a preparative tool, it is more widely used as an analytical tool.
Gel electrophoresis apparatus may be classified into two general types: vertical and horizontal. Both types have been extensively described in the literature including patents. Vertical electrophoresis devices have an advantage in that cleaner separations and smoother flow of the macromolecular mixture to be fractionated are generally possible in vertical apparatus than in horizontal apparatus.
U.S. Pat. No. 4,707,233 shows a representative vertical gel electrophoresis device. The apparatus shown therein comprises an open top container or tank having a basket-like gel slab holder which is placed within the container or tank, a vertical gel slab (or gel cassette) extending through the bottom wall of the gel slab holder, a gasket forming a fluid tight joint between the gel slab and the gel slab holder, and oppositely charged electrodes on opposite sides of the gel slab holder walls, so that electric current must pass through the gel slab. The gel slab comprises pair of parallel plates joined together along their vertical edges by spacers, so that the gel slab is open at the top and bottom but not along the sides, and a sample holder resembling a comb for directing a macromolecular mixture through the gel slab. The comb has a plurality of dividers forming side by side sample wells. This electrophoresis device is not disclosed as being capable of fractionating more than one macromolecular mixture at a time, and would not be suitable for simultaneous resolution of multiple mixtures. The thickness of the dividers in the comb would then cause an uneven electric flux density in the electrical field within the gel slab, with the result that macromolecule streams descending from the sample holder would tend to merge and thus make simultaneous multiple fractionations impossible.
A reusable comb providing a plurality of sample wells and making simultaneous multiple separations possible was developed several years ago by one of the inventors herein and is shown in FIG. 1. FIG. 1 is a top view of this prior art comb. Referring now to FIG. 1, this reusable prior art comb 10 comprises a thin opaque (preferably white) rectangular horizontally extending plastic sheet 12 which is flat before forming, and which as formed comprises a plurality of relatively wide coplanar flat portions 13 alternating with relatively narrow ridges or ribs 14 of square tooth configuration. These ribs 14 extend transversely the entire width of the plastic sheet 12, i.e., from top to bottom. The plastic sheet has two shorter sides of the rectangle. Plastic sheet 12 is laminated to a facing sheet 18 and backing sheet 20, both of which are thin essentially flat transparent rectangular plastic sheets, thinner than sheet 12. The flat main portions 13 of plastic sheet 12 are laminated to the backing sheets 20 while the peaks of ridges 14 are laminated to the facing sheet 18. For the most part facing sheet 18 and backing sheet 20 are parallel and are spaced apart by the height of ridges 14 in plastic sheet 12. However, facing sheet 18 and backing sheet 20 are joined together along parallel side edges 22 thereof, laterally outwardly from the end portions of 16 of plastic sheet 12. This structure forms a plurality of relatively wide vertically extending sample wells, 24, alternating with spacer channels 26 which are much narrower. Sample wells 24 and spacer channels 26 are opened at the top and bottom.
Reusable comb 10 is placed inside a vertical gel cassette 28, shown in broken lines, which is a rectangular prism having a pair of spaced parallel plates forming side walls, and a pair of end walls, providing a vertically extending central opening 30 of rectangular cross section and of width much greater than its thickness. Central opening 30 is open at its top and bottom but is closed along its sides.
The entire interior space 30 of gel cassette 28, including sample wells 24 and spacer channels 26, is filled with a suitable electrophoresis gel e.g., polyacrylamide of gradient concentration, cross linked with a small amount of bis-acrylamide, to achieve the desired fractionation according to molecular weight and/or charge.
Representative dimensions of reusable comb 10 are as follows: width, 16 cm; height (perpendicular to the plane of the paper in FIG. 1), 7 cm; thickness, about 1.5 mm. Representative sheet thicknesses are as follows: opaque plastic sheet 12, 0.2 mm; facing sheet 18 and backing sheet 20, each 0.2 mm. These dimensions may vary, as the thickness of gel cassette 28 and comb 10 are about equal, the former being just enough thicker as to permit insertion and removal of the comb while holding it in place.
A major advantage of the comb 10 over previously known combs or arrangements for vertical electrophoresis devices is that the comb 10 permits simultaneous multiple channel separation of macromolecule mixtures. Separate mixtures may be charged to each of the sample wells 24, and the respective mixtures remain in seperate and distinct lanes are achieved because the electric flux is substantially uniform over the entire interior space 30 of the gel cassette when a comb such as comb 10 illustrated in FIG. 1 is used. The disadvantage of comb 10 is that it is costly to assemble. Lamination of plastic sheet 12 to facing and backing sheets 18 and 20, respectively, must be done by hand, and therefore is the major cost factor. As a result of this high cost combs 10 must be reused. While they are durble and can be used through a number of cycles, they must be cleaned after each cycle, which is difficult because of the small dimensions involved.